Electrocatalytic Palladium Nanoclusters as Versatile Indicators of Bioassays: Rapid Electroanalytical Detection of SARS-CoV-2 by Reverse Transcription Loop-Mediated Isothermal Amplification.

Quantitative polymerase chain reaction (qPCR) is considered the gold standard for pathogen detection. However, improvement is still required, especially regarding the possibilities of decentralization. Apart from other reasons, infectious diseases demand on-site analysis to avoid pathogen spreading and increase treatment efficacy. In this paper, the detection of SARS-CoV-2 is carried out by reverse transcription loop-mediated isothermal amplification, which has the advantage of requiring simple equipment, easily adaptable to decentralized analysis. It is proposed, for the first time, the use of palladium nanoclusters (PdNCs) as indicators of the amplification reaction at end point. The pH of the medium decreases during the reaction and, in turn, a variation in the catalytic activity of PdNCs on the oxygen reduction reaction (ORR) can be electrochemically observed. For the detection, flexible and small-size screen-printed electrodes can be premodified with PdNCs, which together with the use of a simple and small electrochemical equipment would greatly facilitates their integration in field-deployable devices. This would allow a faster detection of SARS-CoV-2 as well as of other future microbial threats after an easy adaptation.

Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label.

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) and laser ablation (LA)-ICP-MS have been complementary employed to develop a comprehensive study of APOE and claudin-1 expression in ARPE-19 cells submitted to a glucose treatment (100 mM, 48 h) that induces oxidative stress conditions. Results were compared with control cells. The determination of the two proteins by ICP-MS was sequentially carried out using specific immunoprobes labelled with IrNCs that offer a huge amplification (1760 ± 90 atoms of Ir on average). A novel sample introduction system, the microFAST Single Cell set-up, was employed for sc-ICP-MS analysis. This introduction system resulted in a cellular transport efficiency of 85 ± 9% for ARPE-19 cells (91 ± 5% using a PtNPs standard). After the proper immunocytochemistry protocol with the specific IrNCs immunoprobes in cell suspensions (sc-ICP-MS), the mass of APOE and claudin-1 in individual ARPE-19 cells was obtained. Average detection limits per cell by sc-ICP-MS were 0.02 fg of APOE and 3 ag of claudin-1. The results of sample analyses obtained by sc-ICP-MS were validated with commercial ELISA kits. The distribution of both target proteins in individual cells (fixated in the chamber wall) was unveiled by LA-ICP-MS. The high amplification provided by the IrNCs immunoprobes allowed the identification of APOE and claudin-1 within individual ARPE-19 cells. High resolution images were obtained using a laser spot of 2 × 2 µm.

Real matrix-matched standards for quantitative bioimaging of cytosolic proteins in individual cells using metal nanoclusters as immunoprobes-label: A case study using laser ablation ICP-MS detection.

The persistent lack of adequate matrix-matched reference materials still hinders the quantitative analysis of elements and biomolecules in biological samples by LA-ICP-MS. This fact is especially critical in cell cultures due to their complex matrix. In this work, we propose a novel matrix-matched calibration strategy, which fully mimics the matrix of cultured cells, by using the same cell line of the sample to create laboratory standards. As a model case, the quantitative imaging of two cytosolic proteins (MT2A and APOE) in individual HRPEsv cells was performed by LA-ICP-MS, both in cells subjected to inflammation with cytokine Interleukin-1α (IL-1α) and controls (CT). A single biomarker strategy using Au nanoclusters (AuNCs) as specific antibody labels was employed for the analysis of the selected proteins in individual cells by LA-ICP-MS. HRPEsv cells supplemented with suspensions containing nude AuNCs was employed to generate single-cell laboratory standards (HRPEsv cells@AuNCs). The preparation and characterization of the single-cell laboratory standards by both ICP-MS and LA-ICP-MS were optimized as well as the data treatment protocol required for obtaining the quantitative distribution of the proteins in individual cells. The mass of APOE and MT2A per cell in CT and IL1α-treated HRPEsv cells analysed by LA-ICP-MS using the proposed matrix-matched calibration were successfully corroborated with commercial ELISA kits. In addition, quantitative real time polymerase chain reaction (qPCR) analyses were performed to study the proteins gene expression.

Gold nanoclusters as elemental label for the sequential quantification of apolipoprotein E and metallothionein 2A in individual human cells of the retinal pigment epithelium using single cell-ICP-MS.

Gold nanoclusters (AuNCs) with a diameter of 1.99 nm on average were synthesized and applied as labels in immunoprobes for the determination of cytosolic proteins in individual human retinal pigment epithelium (HRPEsv) cells by single cell - inductively coupled plasma - mass spectrometry (sc-ICP-MS). For quantitative purposes, the number of gold atoms per immunoprobe (i.e., the amplification factor) was determined; 466 gold atoms on average were obtained. Human metallothioneins (MT), including the 2A isoform (MT2A), and apolipoprotein E (APOE) play an important role under inflammation and oxidation processes in the RPE. The new single biomarker strategy introduced was applied to the sequential determination of MT2A and APOE in HRPEsv cells under pro-inflammatory and control conditions through the development of immunoassays with the corresponding AuNCs immunoprobes and the measurement of the 197Au+ signal by sc-ICP-MS. In addition, 56Fe+ signal was measured as constituent element of HRPEsv cells in order to check the integrity of the cells after the immunoassay and to confirm the number of cell events detected when monitoring the protein label (197Au+). Optimisation of parameters related with the sample preparation for the analysis of cytosolic proteins in intact HRPEsv cells was carried out. The method was successfully applied to the determination of both proteins in control cells and cells treated with the recombinant human interleukin-1α. Quantitative results obtained per cell for the average protein amounts of APOE and MT2A using the sc-ICP-MS procedure were corroborated with commercial ELISA kits.

Iridium nanoclusters as high sensitive-tunable elemental labels for immunoassays: Determination of IgE and APOE in aqueous humor by inductively coupled plasma-mass spectrometry.

Iridium nanoclusters (IrNCs) stabilized with citrate were synthesized and then the ligand was exchanged by lipoic acid (LA). The IrNCs@LA were bioconjugated through carbodiimide chemistry with specific antibodies to prepare IrNCs-labelled immunoprobes. The IrNCs-immunoprobes were employed in competitive immunoassays for immunoglobulin E (IgE) and apolipoprotein E (APOE) determination by detecting the iridium through inductively coupled plasma - mass spectrometry (ICP-MS). The IrNCs@LA have a 1.89 nm diameter at average and each NC contains 250 Ir atoms. Labelling of specific antibodies with IrNCs was optimized in terms of recognition capabilities and signal amplification by ICP-MS. Amplification and detection limits can be tuned by selecting the IrNCs:Ab molar ratio. An immunoprobe prepared by mixing a 10:1 IrNC:Ab molar ratio was selected for the determination of IgE and APOE in aqueous humor, achieving a signal amplification of 1760 iridium atoms per molecule of the sought protein and limits of detection in the tens of pg mL-1 of protein. The IrNCs-immunoprobes were evaluated for IgE determination in serum samples as well as for IgE and APOE in aqueous humor (from controls subjects and patients affected by primary open angle glaucoma) by ICP-MS, being required just sample dilution as pre-treatment. Results were corroborated with commercial ELISA kits.

Multiplex bioimaging of proteins-related to neurodegenerative diseases in eye sections by laser ablation - Inductively coupled plasma - Mass spectrometry using metal nanoclusters as labels.

Simultaneous determination of proteins with micrometric resolution is a significant challenge. In this study, laser ablation (LA) inductively coupled plasma - mass spectrometry (ICP-MS) was employed to quantify the distribution of proteins associated to the eye disease age-related macular degeneration (AMD) using antibodies labelled with three different metal nanoclusters (MNCs). PtNCs, AuNCs and AgNCs contain hundreds of metal atoms and were used to detect metallothionein 1/2 (MT1/2), complement factor H (CFH) and amyloid precursor protein (APP) in retina, ciliary body, retinal pigment epithelium (RPE), choroid and sclera from human cadaveric eye sections. First, the labelling of MNCs bioconjugated primary antibodies (Ab) was optimised following an immunolabelling protocol to avoid the non-specific interaction of MNCs with the tissue. Then, the LA and ICP-MS conditions were studied to obtain high-resolution images for the simultaneous detection of the three labels at the same tissue section. A significant signal amplification was found when using AuNCs, AgNCs and PtNCs labelled Ab of 310, 723 and 1194 respectively. After the characterisation of MNCs labelled immunoprobes, the Ab labelling was used for determination of MT1/2, CFH and APP in the RPE-choroid-sclera, where accumulation of extracellular deposits related to AMD was observed. Experimental results suggest that this method is fully suitable for the simultaneous detection of at least three different proteins.

Nanoparticles as labels of specific-recognition reactions for the determination of biomolecules by inductively coupled plasma-mass spectrometry.

Inductively coupled plasma - mass spectrometry (ICP-MS) is an essential tool for quantitative ultratrace elemental and isotopic determinations in the biological and biomedical fields. In addition, sensitive and multiplexed quantification of target biomolecules in biological fluids and single-cells by ICP-MS can be carried out using metal elements or their isotopes as labels of immunoreaction and hybridization specific-recognition reactions. Following similar strategies, the bioimaging of biomolecules in tissues and single-cells by laser ablation (LA)-ICP-MS can be also achieved. Sensitivity can be further increased by resorting to amplification strategies based on the use of labels containing several atoms of a given elemental (or isotopic) reporter, such as inorganic nanoparticles (NPs). In this review, we intend to highlight the progress achieved in this active research area. Following the introduction, a short report of the characteristics of the most relevant NPs used as labels of specific-recognition reactions for ICP-MS detection, as well as the most common labelling routes are given. Then, the applications of NPs-labelled detection probes used in combination with ICP-MS (either with liquid nebulization or LA sampling) for identification and determination of proteins and oligonucleotides in biological samples are thoroughly reviewed. In the conclusions section, the challenges faced and the expected advances in this topic are underlined.

Bimodal determination of immunoglobulin E by fluorometry and ICP-MS by using platinum nanoclusters as a label in an immunoassay.

The authors describe the use of platinum nanoclusters (PtNCs) as bimodal labels in a competitive immunoassay for immunoglobulin E (IgE). Both fluorometry and inductively coupled plasma - mass spectrometry (ICP-MS) are used. Optimization of the PtNCs synthesis using lipoic acid as ligand was carried out. The time for synthesis and the effect of NaOH added to the PtNCs precursor mixture was optimized with the aim to obtain PtNCs with strong red fluorescence and low size dispersity. Maximal fluorescence was obtained at excitation/emission wavelengths of 455/620 nm. The average diameter (1.5 nm) and crystal structure (face-centered cubic structure) of the PtNCs were determined by HR-TEM. It was calculated that each PtNC contains 116 Pt atoms at average. Labelling of the antibody (Ab) against IgE with PtNCs was optimized in terms of recognition capabilities and fluorescence intensity. A molar ratio (Ab:PtNCs) of 1:11 is found to be best. A competitive immunoassay for IgE was developed and detection was carried out by using both ICP-MS (by measuring 195Pt) and fluorometry. The limit of detection (LOD) of the fluoroimmunoassay is 0.6 ng mL-1 of IgE. The LOD of the ICP-MS method is as low as 0.08 ng mL-1. The method was evaluated by analyzing four (spiked) serum samples by ICP-MS. No sample pretreatment excepting dilution is needed. Results compared favorably with those obtained by a commercial ELISA kit. Graphical abstract Schematic representation of the bimodal quantification (fluorescence and ICP-MS) of immunoglobulin E (Ig E) in human serum using antibody against human Ig E, labelled with several platinum nanoclusters (NCs) as immunoprobe. Elemental mass spectrometry (MS) allows high amplification of the signal because of the high number of platinum atoms per nanocluster (~116 Pt/NC).

Quantitative mapping of specific proteins in biological tissues by laser ablation-ICP-MS using exogenous labels: aspects to be considered.

Laser ablation (LA) coupled with inductively coupled plasma mass spectrometry (ICP-MS) is a versatile tool for direct trace elemental and isotopic analysis of solids. The development of new strategies for quantitative elemental mapping of biological tissues is one of the growing research areas in LA-ICP-MS. On the other hand, the latest advances are related to obtaining not only the elemental distribution of heteroatoms but also molecular information. In this vein, mapping of specific proteins in biological tissues can be done with LA-ICP-MS by use of metal-labelled immunoprobes. However, although LA-ICP-MS is, in principle, a quantitative technique, critical requirements should be met for absolute quantification of protein distribution. In this review, progress based on the use of metal-labelled antibodies for LA-ICP-MS mapping of specific proteins is reported. Critical requirements to obtain absolute quantitative mapping of specific proteins by LA-ICP-MS are highlighted. Additionally, illustrative examples of the advances made so far with LA-ICP-MS are provided. Graphical abstract In the proposed critical review, last advances based on the use of metal-labelled antibodies and critical requirements for LA-ICP-MS quantitative mapping of specific proteins are tackled.